1.1 Field of the Invention
The present invention relates generally to the fields of molecular biology and virology, and in particular, to genetic expression cassettes, and vector comprising them useful for the delivery of nucleic acid segments encoding selected therapeutic constructs (including for example, peptides, polypeptides, ribozymes, and catalytic RNA molecules), to selected cells and tissues of vertebrate animals. In particular, these genetic constructs are useful in the development of gene therapy vectors, including for example, HSV, AV, and AAV vectors, for the treatment of mammalian, and in particular, human diseases, disorders, and dysfunctions. The disclosed compositions may be utilized in a variety of investigative, diagnostic and therapeutic regimens, including the prevention and treatment of a variety of human diseases. Methods and compositions are provided for preparing viral vector compositions comprising these genetic expression cassettes for use in the preparation of medicaments useful in central and targeted gene therapy of diseases, disorders, and dysfunctions in an animal, and in humans in particular.
1.2 Description of the Related Art
Currently, viral vectors show the greatest efficiency in gene transfer (reviewed in Anderson, 1998; Verma and Somia, Nature, 1997). For correction of genetic diseases such that persistent gene expression is required, herpesvirus, retrovirus, lentivirus, adenovirus, or AAV based vectors are desirable due to the integrating nature of the viral life cycle.
In considering transgene expression, there are many known situations where a transferred gene(s) is capable of a short period of expression however followed by a decline to undetectable levels without the loss of the expression construct. These expression constructs may sustain transgene expression for periods of time up to 2 weeks and on rare occasions 2 months (Palmer et al., 2000). Unfortunately, despite claims of sustained expression up to 2 months, the over-ruling factor is that one can anticipate an eventual decline of transcript levels often to near zero levels. As a result, this presents an additional variable to transgene expression; the predictability or probability of transgene expression. For the purposes of gene therapy, transgene expression kinetics must be predictable to achieve safe and reliable therapeutic effects.
The mechanisms responsible for transcript loss have been attributed to elaborate defense mechanisms used by eukaryotic cells to protect both the structure of their genomes and to oppose expression of abnormal transcription units (Bestor, 2000). These mechanisms include, but are not limited to, DNA methylation, multi-copy repeat-induced transgene silencing, post-transcriptional gene silencing (PTGS) mediated by RNAi, position effects that impose histone methylation/deacetylation. These host defense mechanisms represent a formidable barrier to many forms of gene therapy. Current gene therapy applications often depend on a construct or recombinant virus with the ability to express an agent of interest (protein or RNA) in a particular tissue. However, cells can detect alterations within their genome due to multi-copy transgene insertions or to abnormal transcripts and elicit a strong and heritable silencing effect. A common example of multi-copy transgene silencing is in the generation of transgenic animals. It had previously been found that transgene copy number was inversely proportional to the level of gene expression in some lines of transgenic mice. It is thought that end-to-end ligation of the expression construct and/or homologous recombination between construct molecules generates transgene concatemers (often 5-50 copies) that integrate at a single site within the genome (Dobie et al., 1997). Unfortunately, the tandem repeats appear to contribute to a phenomenon similar to position effect varigation (PEV). PEV may be the result of position-dependent inactivation of the expression construct mediated by the surrounding heterochromatin environment and results in the heritable maintenance of the transcription “off” state (Dobie et al., 1997).